Pancreatic (pro)enzymes treatment suppresses BXPC-3 pancreatic Cancer Stem Cell subpopulation and impairs tumour engrafting.

Cancer stem cells (CSCs) subpopulation within the tumour is responsible for metastasis and cancer relapse. Here we investigate in vitro and in vivo the effects of a pancreatic (pro)enzyme mixture composed of Chymotrypsinogen and Trypsinogen (PRP) on CSCs derived from a human pancreatic cell line, BxPC3. Exposure of pancreatic CSCs spheres to PRP resulted in a significant decrease of ALDEFLUOR and specific pancreatic CSC markers (CD 326, CD 44 and CxCR4) signal tested by flow cytometry, further CSCs markers expression was also analyzed by western and immunofluorescence assays. PRP also inhibits primary and secondary sphere formation. Three RT2 Profiler PCR Arrays were used to study gene expression regulation after PRP treatment and resulted in, (i) epithelial-mesenchymal transition (EMT) inhibition; (ii) CSCs related genes suppression; (iii) enhanced expression of tumour suppressor genes; (iv) downregulation of migration and metastasis genes and (v) regulation of MAP Kinase Signalling Pathway. Finally, in vivo anti-tumor xenograft studies demonstrated high anti-tumour efficacy of PRP against tumours induced by BxPC3 human pancreatic CSCs. PRP impaired engrafting of pancreatic CSC's tumours in nude mice and displayed an antigrowth effect toward initiated xenografts. We concluded that (pro)enzymes treatment is a valuable strategy to suppress the CSC population in solid pancreatic tumours.

Effects of trypsinization and mineralization on intrasynovial tendon allograft healing to bone.

The purpose of the current study was to develop a novel technology to enhance tendon-to-bone interface healing by trypsinizing and mineralizing (TM) an intrasynovial tendon allograft in a rabbit bone tunnel model. Eight rabbit flexor digitorum profundus (FDP) tendons were used to optimize the trypsinization process. An additional 24 FDP tendons were stratified into control and TM groups; in each group, 4 tendons were used for in vitro evaluation of TM and 8 were transplanted into proximal tibial bone tunnels in rabbits. The samples were evaluated histologically and with mechanical testing at postoperative week 8. Maximum failure strength and linear stiffness were not significantly different between the control and TM tendons. A thin fibrous band of scar tissue formed at the graft-to-bone interface in the control group. However, only the TM group showed obvious new bone formation inside the tendon graft and a visible fibrocartilage layer at the bone tunnel entrance. This study is the first to explore effects of TM on the intrasynovial allograft healing to a bone tunnel. TM showed beneficial effects on chondrogenesis, osteogenesis, and integration of the intrasynovial tendon graft, but mechanical strength was the same as the control tendons in this short-term in vivo study.

In vitro treatment of carcinoma cell lines with pancreatic (pro)enzymes suppresses the EMT programme and promotes cell differentiation.

BACKGROUND: Previous research has suggested a putative utility of pancreatic (pro)enzymes in cancer treatment. The aim of the present study was to investigate the in vitro effects of a mixture of two pancreatic pro-enzymes, i.e., Chymotrypsinogen and Trypsinogen, and the enzyme Amylase on three human cancer cell lines, i.e., OE33 (derived from an oesophageal carcinoma), Panc1 (derived from a pancreatic carcinoma) and Caco-2 (derived from a colon carcinoma). RESULTS: After treatment of the three cancer cell lines with different doses of the (pro)enzymes for up to 7 days, we observed (i) growth inhibition in a dose-dependent manner, (ii) enhanced expression of ß-catenin and E-cadherin and decreased expression of several epithelial-mesenchymal transition (EMT)-associated genes, such as Vimentin, Snail and Slug, (iii) differentiation of Caco-2 cells, including the appearance of cell-specific differentiated structures such as microvilli and tight junctions, the acquisition of a more regular polygonal morphology, and an increased expression of the intestinal differentiation markers alkaline phosphatase and cytokeratin 8, and (iv) differentiation of Panc1 cells, including the formation of cell aggregates, an increment on lamellar bodies and an increased expression of the pancreatic differentiation markers glucagon and insulin. CONCLUSIONS: Our results show that the treatment of three different human cancer cell lines with pancreatic (pro)enzymes results in an enhancement of cell adhesion, an attenuation of several EMT-associated markers, and an increase in the expression of several differentiation-associated markers, suggesting the acquisition of a less malignant phenotype and a decrease in proliferative capacity due to lineage-specific cellular differentiation.

Trypsin-2 degrades human type II collagen and is expressed and activated in mesenchymally transformed rheumatoid arthritis synovitis tissue.

It has traditionally been believed that only the human collagenases (matrix metalloproteinase-1, -8, and -13) are capable of initiating the degradation of collagens. Here, we show that human trypsin-2 is also capable of cleaving the triple helix of human cartilage collagen type II. We purified human trypsin-2 and tumor-associated trypsin inhibitor by affinity chromatography whereas collagen type II was purified from cartilage extracts using pepsin digestion and salt precipitation. Degradation of type II collagen and gelatin by trypsin-2 was demonstrated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis, zymography, and mass spectrometry, and tumor-associated trypsin inhibitor specifically inhibited this degradation. Although human trypsin-2 efficiently digested type II collagen, bovine trypsin did not. Furthermore, immunohistochemical staining detected trypsin-2 in the fibroblast-like synovial lining and in stromal cells of human rheumatoid arthritis synovial membrane. These findings were confirmed by reverse transcriptase-polymerase chain reaction and nucleotide sequencing. Trypsin-2 alone and complexed with alpha(1)-proteinase inhibitor were also detected in the synovial fluid of affected joints by time-resolved immunofluorometric assay, suggesting that trypsin-2 is activated locally. These results are the first to assess the ability of human trypsin to cleave human type II collagen. Thus, trypsin-2 and its regulators should be further studied for use as markers of prognosis and disease activity in rheumatoid arthritis.

Proenzyme therapy of cancer.

Proteases and their inhibitors have long been investigated in numerous tumor systems, and at the tumor growing front, their balance has been universally found to be shifted towards higher proteolytic activities. However, out of many promising serine and metalloproteinase inhibitors, none are included in cancer treatment regimens at present. The current search for active antiproteolytic compounds is in contrast to the classical approach developed by John Beard, who suggested treating advanced cancer by fresh pancreatic extracts whose antitumor activity was based on their proteolytic potential. We followed John Beard's recommendations by using purified pancreatic proenzymes/enzymes, trypsinogen/trypsin (TG/TR), chymotrypsinogen/chymotrypsin (CG/CH) and amylase (AM). The mixture of these enzymatic activities produces potent antimetastatic and antitumor effects in cellular, animal and human systems. The treatment of cultured tumor cells with TR and CH at nanomolar [corrected] concentrations, comparable to those achieved in the blood of the patients, causes complete arrest of the directional movement of metastatic cells. Conversely, the same treatment of normal cells results in enhanced motility and an accelerated closure of the gap created in cell monolayers. Further, treatment of cells with serine proteases results in the formation of cellular 3-dimensional structures such as lamellae, cell streams and aggregates. In some cell types, the aggregates are compacted via cadherin-based cell-cell communication systems and form compact spheroids. In the highly metastatic cells with lower cadherin expression, the ability to form spheroids also diminishes. Tumor cells unable to form spheroids when treated with proteases are subject to elimination by apoptosis. In contrast, a large proportion of cells that form spheroids remain viable, although they are metabolically suppressed. Protease-treated tumor cells contain a disrupted actin cytoskeleton and exhibit a loss of front-to-back polarity. We hypothesize that the provision of zymogens, rather than the enzymes, was of crucial importance to the clinical effectiveness in the human trials conducted by Beard and his co-workers. The precursor nature of the active enzymes may offer protection against numerous serpins present in the tissues and blood. Experimental evidence supports the assertion that the conversion from proenzyme to enzyme occurs selectively on the surface of the tumor cells, but not on normal cells. We believe that this selectivity of activation is responsible for the antitumor/antimetastatic effect of proenzyme therapy and low toxicity to normal cells or tumor host. Elevated levels of endostatin and angiostatin appear in the blood of TG/CG/AM-treated tumor-bearing mice, but not in tumor mice treated with the vehicle alone or in proenzyme-treated tumor-free mice. These findings support the conclusion that proteolysis is the active mechanism of the proenzyme treatment. Future studies will focus on the molecular mechanisms of the proenzyme therapy including the identification of molecular target(s) on the tumor cells. In conclusion, we have discovered that proenzyme therapy, mandated first by John Beard nearly one hundred years ago, shows remarkable selective effects that result in growth inhibition of tumor cells with metastatic potential.

Genetic factors in pancreatitis.

The understanding of pathogenesis of acute and chronic pancreatitis has benefited from the progress made in genetic investigations. The discoveries of the gain of function mutations of cationic trypsinogen gene (PRSS1) and the loss of function mutations of pancreatic secretory trypsin inhibitor (SPINK 1) or other potential defects in genes that regulate pancreatic secretory function or modulate inflammatory response to pancreatic injury has changed our current concepts on the pathogenesis of pancreatitis. Genetic factors play an important role in the susceptibility to pancreatic injury, severity and evolution of inflammatory process, leading in some cases to chronic inflammation and/or fibrosis. Acute pancreatitis is viewed as an event and chronic pancreatitis as a process, sequentially linked, reflecting a complex interaction between genetic and environmental factors.

S100 family members and trypsinogens are predictors of distant metastasis and survival in early-stage non-small cell lung cancer.

Distant metastasis is the predominant cause of death in early-stage non-small cell lung cancer (NSCLC). Currently, it is impossible to predict the occurrence of metastasis at early stages and thereby separate patients who could be cured by surgical resection alone from patients who would benefit from additional chemotherapy. In this study, we applied a comparative microarray approach to identify gene expression differences between early-stage NSCLC patients whose cancer ultimately did or did not metastasize during the course of their disease. Transcriptional profiling of 82 microarrays from two patient groups revealed differential expression of several gene families including known predictors of metastasis (e.g., matrix metalloproteinases). In addition, we found S100P, S100A2, trypsinogen C (TRY6), and trypsinogen IVb (PRSS3) to be overexpressed in tumors that metastasized during the course of the disease. In a third group of 42 patients, we confirmed the induction of S100 proteins and trypsinogens in metastasizing tumors and its significant correlation with survival by real-time quantitative reverse transcription-PCR. Overexpression of S100A2, S100P, or PRSS3 in NSCLC cell cultures led to increased transendothelial migration, corroborating the role of S100A2, S100P, and PRSS3 in the metastatic process. Taken together, we provide evidence that expression of S100 proteins and trypsinogens is associated with metastasis and predicts survival in early stages of NSCLC. For the first time, this implicates a role of S100 proteins and trypsinogens in the metastatic process of early-stage NSCLC.

Tumor-associated trypsinogen-2 (trypsinogen-2) activates procollagenases (MMP-1, -8, -13) and stromelysin-1 (MMP-3) and degrades type I collagen.

A critical step in cancer growth and metastasis is the dissolution of the extracellular matrix surrounding the malignant tumor, which leads to tumor cell invasion and dissemination. Type I collagen degradation involves the initial action of collagenolytic matrix metalloproteinases (MMP-1, -8, and -13) activated by MMP-3 (stromelysin-1). The role of interactive matrix serine proteinases (MSPs), including tumor-associated trypsinogens, has been unclear in collagenolysis. Now, we provide evidence that the major isoenzyme of human tumor-associated trypsinogens, trypsin-2, can directly activate three collagenolytic proMMPs as well as proMMP-3. These proMMP activations are inhibited by tumor-associated trypsin inhibitor (TATI). Furthermore, we demonstrate that trypsin-2 efficiently degrades native soluble type I collagen, which can be inhibited by TATI. However, cell culture studies showed that trypsin-2 transfection into the HSC-3 cell line did not result in MMP-1, -3, -8, and -13 activation but affected MMP-3 and -8 production at the protein level. These findings indicate that human trypsin-2 can be regarded as a potent tumor-associated matrix serine protease capable of being the initial activator of the collagenolytic MMP activation network as well as directly attacking type I collagen.

Trypsin and activation of circulating trypsinogen contribute to pancreatitis-associated lung injury.

Pancreatic proteases are secreted in acute pancreatitis, but their contribution to associated lung injury is unclear. Applying models of mild edematous (intravenous caerulein) and severe necrotizing (intraductal glycodeoxycholic acid) pancreatitis in rats, we showed that both trypsinogen and trypsin concentrations in peripheral blood, as well as lung injury, correlate with the severity of the disease. To isolate the potential contribution of proteases to lung injury, trypsin or trypsinogen was injected into healthy rats or trypsinogen secreted in caerulein pancreatitis was activated by intravenous enterokinase. Pulmonary injury induced by protease infusions was dose dependent and was ameliorated by neutrophil depletion. Trypsinogen activation worsened lung injury in mild pancreatitis. In vitro incubation of leukocytes with trypsinogen showed that stimulated leukocytes can convert trypsinogen to trypsin. In conclusion, this study demonstrates that the occurrence and severity of pancreatitis-associated lung injury (PALI) corresponds to the levels of circulating trypsinogen and its activation to trypsin. Neutrophils are involved in both protease activation and development of pulmonary injury.

[Trypsinogen as a modifier of peptidergic influences on the secretion of the gastric and pancreatic glands]. / Tripsinogen kak modifikator peptidergicheskikh vliianii na sekretsiiu zheludochnykh i podzheludochnoi zhelez.

I.v. administration of trypsinogen potentiated the pentagastrin-induced secretion of gastric glands and reduced the leu-enkephalin effect upon them in the fistula dogs. Intraperitoneal administration of trypsinogen enhanced the pepsinogen synthesis by the gastric glands, did not change the hydrolases activity in pancreas homogenate in rats.

Identification of the site of phosphorylation on the osmosensor, EnvZ, of Escherichia coli.

EnvZ is a membrane-bound histidine kinase that functions as an osmotic sensor capable of phosphorylating the regulator protein OmpR in Escherichia coli. To characterize the site of phosphorylation biochemically, we overexpressed a 36-kDa truncated EnvZ protein (Glu-106 to Gly-450) that formed inclusion bodies in the cell. After solubilization, the inclusion body form of EnvZ was cleaved into two major fragments with molecular weights of 25,000 and 10,000. The 25-kDa fragment, EnvZc, was purified and found to exist as a dimer. N-terminal sequence analysis established that cleavage had occurred at Arg-214, indicating that EnvZc contained most of the cytoplasmic domain of EnvZ. After labeling EnvZc with [gamma-32P]ATP, the protein was proteolytically digested, and the resulting peptides were separated by reverse phase chromatography using high performance liquid chromatography. One major radioactive peptide containing greater than 90% of the recovered peptide-associated radioactivity was isolated. Amino acid analysis of this purified peptide indicated that the composition was consistent with a peptide that contained His-243. The amino acid sequence of this peptide was determined to be MAGVSHDLRTP (residues 238-248). These results indicate that His-243 is the major site of phosphorylation on EnvZ and represents the first biochemical characterization of the site of phosphorylation of a membrane histidine kinase of the two-component regulatory family of molecules in bacteria.

[Inhibition of pancreatic secretion by trypsin and trypsinogen]. / Tormozhenie sekretsii podzheludochnoi zhelezy tripsinom i tripsinogenom.

In dogs, trypsinogen inhibited the secretion of proteases more selectively and for a longer period than trypsin, whereas trypsin reduced tryptic activity of the blood to a lesser extent than trypsinogen. In contrast to trypsinogen, trypsin increased renal excretion of amylase and lipase. The difference between the effects may be due to the action of the trypsinogen hexapeptide formed during its turning into trypsin. Dynamic self-regulation of pancreatic secretion seems to depend on the ratio of trypsin, trypsinogen, its hexapeptide and products of alimentary proteins hydrolysis in duodenal contents.

Dose-related inhibition of gastric secretion by intraduodenal trypsinogen in dogs.

It has been shown previously that trypsinogen and its activation peptide but not trypsin decreased gastric secretion. The purpose of this work was to study the dose-action relation between the intraduodenal infusion of trypsinogen and gastric secretion. Three dogs provided with gastric and duodenal Thomas fistulae were stimulated by continuous i.v. perfusion of porcine gastrin I-II (6 microgram kg-1 h-1). Pancreatic juice was diverted to the exterior and gastric secretion was collected. Upon reaching a gastric secretory plateau, porcine trypsinogen was infused intraduodenally at doses of 5, 10, 20, 40, 80 and 160 mg. Each test was continued for a further 60 min. Control was made with isotonic saline. There was a dose-related inhibition of the gastrin-stimulated gastric acid output. This inhibition reached a maximum of 50% with 40 mg of intraduodenal trypsinogen, showing no increase with higher doses.

[Anabolic effects of parenterally administered gastric gland hydrolases]. / Anabolicheskie vliianiia parenteral'no vvodimykh gidrolaz pishchevaritel'nykh zhelez.

In rats, i.v. administration of amylase, pepsinogen, and tripsinogen in microdoses increased the incretion of radiomethyonin from the blood into the tissue proteins of some organs. The incretion of pepsinogen stimulated the protein-production function of pancreas, tripsinogen stimulated the function of stomach glands, amylase--the function of the liver and the small intestine mucosa. The labeled pepsinogen 125I concentrated mainly in secretory organs.

Influence of biliary stasis on enterokinase activity in the rat.

After 48 h of bile duct ligation, enterokinase activity in rat mucosa was significantly lowered in comparison with controls or rats with bile fistula. Rats with bile fistula were similar to controls. Sucrase levels were similar in all groups. Without endogenous trypsinogen, duodenal perfusion with a trypsinogen-containing solution led to more conversion to trypsin by controls. When their own pancreatic secretion was the source of trypsinogen, trypsin was much higher in perfusate from bile-ligated rats. Solubilized enterokinase was also higher in this group. These results indicate that bile stasis leads to decreased mucosal enterokinase activity and increased pancreatic function. The latter may have caused accelerated loss of enterokinase into the lumen, leading to lower mucosal levels.

[Effect of trypsinogen on the secretion of the gastric glands]. / Vliianie tripsinogena na sekretsiiu zheludochnykh zhelez.

In chronic experiments on dogs, i. v. administration of trypsinogen (5 mg) raised enzyme--secreting activity of the gastric glands. An increase in pepsinogen secretion by the gastric glands was noted also in acute experiments early after ligation of pancreatic ducts when tryptic activity of the blood was rising. The activating effect of trypsinogen was also revealed on the rat stomach in vitro.

Pancreatic extract and the intestinal uptake of vitamin B12. II. Inhibitory effect of trypsin and trypsinogen.

Pancreatic extract (PE) contained small-molecular, thermo-stable as well as macro-molecular, thermo-labile factors capable of reducing the uptake of 57CoB12 bound to rat intrinsic factor by perfused rat intestinal segments (p less than 0.01 and p less than 0.01). Neither non-radioactive vitamin B12 nor non-pacreatic protein reduced the 57CoB12-uptake (p greater than 0.5 and p greater than 0.1) Crystalline trypsin and trypsinogen, but not chymotrypsin, also inhibited the uptake (p less than 0.05, p less than 0.02 and p greater than 0.05). The tryptic inhibition was abolished by soybean trypsin inhibitor (p greater than 0.05).